1. Field of the Invention
The present invention relates to an eliminator of adenosine-5'-triphosphate (referred to hereinafter as ATP), a process for eliminating ATP therewith, a reagent for measuring biological cells and a process for measuring biological cells therewith.
More particularly, the present invention relates to the evaluation of the biological contamination of samples such as foods and drinks, or the half-products or materials thereof by treating the samples with the ATP eliminator and then measuring ATP in contaminant microorganism cells contained in the samples by the bioluminescence method.
2. Description of Related Art
The measurement of biological cells such as Escherichia coli, yeast, and lactic acid bacteria is very important in the fields including food hygiene, biotechnology, clinical laboratory tests, medicine, super pure water and environment.
Biological cells are generally measured by severals methods such as microscopic instrumentation with a hematocytometer (microscopic method), turbidimetry, gravimetry, packed volume measuring method, and colony counting method (referred to hereinafter as pour culture method).
The microscopic method, the turbidimetry, the gravimetry and the packed volume measuring method, however, have disadvantages of low sensitivity or inability to distinguish unviable cells and viable cells, while the pour culture method is not suitable for the cases which are desired to obtain the result quickly since it requires the culture of cells and thus usually requires a period of time for one or more days.
The counting of cells in the aforementioned fields requires the rapid measurement with high sensitivity; the test of microbial contamination of products is essentially required for their shipping in the field of food hygiene. The test has been conventionally carried out by the pour culture method, which requires a period of time for one or more days, so that the products must be stored in a warehouse until the products are guaranteed by the test. This causes not only a problem in the point of logistic efficiency, but also the risk increased of microbial contamination in food products such as milk with the prolonged time of storage. In addition, microorganisms which become an issue for the contamination of foods are generally in a low level, so that a test with high sensitivity is required.
The method for measuring the level of microorganisms which satisfies the requirements described above includes the bioluminescence method of measuring ATP which is present in all of viable microorganisms. This is a method for measuring cells by placing a sample containing the cells into contact with an extraction reagent containing surfactants, trichloroacetic acid (TCA), a Tris-buffer, ethanol or a lytic enzyme to release intracellular ATP out of the cells, placing the ATP into contact with a luminescent reagent which contains luciferin as a substrate of luminescence in a firefly and the enzyme luciferase for producing bioluminescence as a result of the enzyme reaction of luciferin, luciferase and ATP, and measuring the amount of luminescence produced for the determination of the intracellular ATP.
However, ATP is originally present in varying amounts in all of biological cells including not only microorganisms but also unicellular organisms as well as animal and plant tissues in which it is present as the so-called somatic cells. Furthermore, ATP is also present in the free form in the surroundings of biological cells.
Thus, even if it is intended to detect ATP which is contained only in a certain biological cell from a sample containing biological cells, ATP in the biological cells is detected together with free ATP in the neighborhood of the biological cells. In other words, if ATP is intended to be used as an index for the measurement of the biological cells, the free ATP other than that in the biological cells described above is measured as the background luminescence level (noise) together with ATP in the biological cells, and thus such a measurement has a defect of lowering the detection sensitivity of ATP.
As the techniques for eliminating the free ATP, several methods are currently employed including the membrane filter method in which ATP is eliminated from the sample through a membrane filter, the centrifugation method in which it is eliminated by centrifugation, the method for eliminating ATP with an enzyme such as apyrase, adenosine triphosphatase (ATPase), hexokinase, or ATP pyrophosphatase (referred to hereinafter as enzymatic method) (Monthly Food Chemical, SHOKUHIN KAGAKU SHINBUN-SHA, May, 1995, pp. 55-63; Japanese Patent Laid-Open Publication No. 65800/1990; U.S. Pat. No. 5,316,907; Analitical Biochemistry, 218, 20-25, 1994; Bulletin of the Japanese Society of Scientific Fisheries, 52 (9), 1695, 1986; PROC. N.A.S. Vol 52, 1580-1586, 1964; PCT WO 94/28169; and Marine Ecology-Progress Series, 13, 305-309, 1983).
The membrane filter method has however disadvantages in that it is complicated in operation, inferior in filtration ability and insufficient of the ATP eliminating effect.
The centrifugation method also has a disadvantage in that it is complicated in operation and solid components in samples are sedimented and separated together with bacteria, so that it cannot realize the measurement at high precision.
Furthermore, the enzymatic method has also a disadvantage in that it is difficult to lower the background due to the sample, and the ATP eliminating effect remains in unsatisfactory levels.